Prion diseases or transmissible spongiform encephalopathies are infectious neurodegenerative diseases of humans and animals. A major feature of prion diseases is the refolding and aggregation of a normal host protein, prion protein (PrP), into a disease-associated protease-resistant form (PrPres) which may contribute to brain damage. Previously we studied the formation of the amyloid and non-amyloid forms of disease-associated protease-resistant prion protein (PrPres) in scrapie-infected mice. After scrapie infection in transgenic mice expressing anchorless PrP, amyloid is deposited in brain mainly in the perivascular pattern similar to cerebral amyloid angiopathy (CAA) seen in Alzheimers disease and some genetic brain diseases in humans. In contrast to the results seen in transgenic mice, infection of mice expressing anchored PrP gave rise to non-amyloid PrPres which was not associated with blood vessels or with ISF tracers. In FY14 we studied the spread of prion infection within the central nervous system after targeted microinjection of a 0.5 microliter amount of scrapie brain homogenate into a particlar brain region (striatum) of recipient transgenic mice. By euthanizing mice at various times from 20 to 360 days post-injection we were able to follow to localization of the spread of disease-associated PrP using conventional immunohistochemistry. In these studies, we discovered an important role for the brain interstitial fluid (ISF) in spread of prion infectivity in brain. Instead of following the neuronal circuitry as has been seen in normal animals expressing membrane-anchored PrP, in transgenic mice expressing only anchorless PrP, the infection appeared to proceed slowly along the outside of blood vessels in the same areas where the ISF is known to flow. This finding demonstrated a new feature of prion infection related to anchorless PrP. Since non-transgenic mice and other species also are known to express some amount of anchorless PrP, this finding may also have relevance for spread in these non-transgenic mice as well as in transgenic mice. In FY14 we also participated in a proteomics study in collaboration with Drs. Priola and Moore in LPVD, where levels of proteins with altered expression in scrapie infected mice were determined using mass spectrometry. Scrapie-infected mice expressing either anchorless PrP or anchored PRP were compared and were found to differ in some aspects of protein dysregulation associated to the amyloid and non-amyloid types of prion disease associated with these two type of mice.